It is desirable for both research and clinical reasons to monitor analyse and spatially characterise cellular and sub-cellular patterns. Such patterns may, for example, be monitored, analysed and/or characterised using histological staining and immunohistochemistry.
Immunohistochemistry (IHC) refers to the process of detecting antigens (e.g. proteins) in cells of a tissue sample by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumours, since specific molecular markers are characteristic of particular cellular events such as proliferation or cell death. IHC is also widely used in basic research to understand the distribution and localisation of biomarkers and differentially expressed proteins in different parts of a biological tissue.
Visualising an antibody-antigen interaction can be accomplished in a number of ways. Most commonly, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction. Alternatively, the antibody can be tagged to a fluorophore, such as fluorescein or rhodamine.
Researchers regularly use IHC markers in conjunction with an imaging technique to make diagnostic and prognostic decisions in a range of diseases. To do this, they visually inspect the images and draw on their training and experience to identify the presence or absence of a particular type of cell or sub-cellular structure and the spatial arrangement thereof. This identification is based on visual recognition of particular patterns of distinct cells which are indicative of specific or general cellular processes. It is therefore a subjective technique, completely reliant on the human eye, and as an image is easily affected by factors such as strong background staining, weak target antigen staining and auto fluorescence, decisions vary between researchers and clinicians and except in advanced research centres quantification of an imaged tissue sample is typically limited to counting cells.
Additionally even with sophisticated analysis and visualisation tools, it is difficult for even expert clinicians and/or researchers to interpret images including a plurality of biomarkers. For example, analysis becomes particularly complex once the number of biomarkers exceeds 3 or 4, since as the number of markers increases, the ability of the human eye to distinguish between the different markers decreases. The image characterisation is hampered by stains which may be occurring concurrently in cells close together, interacting or expressed by the same cell.
An alternative technique for this analysis of multiple biomarkers is tissue microarray (TMA). TMAs consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis. In the technique, a hollow needle is used to transfer tissue cores as small as 0.6 mm in diameter from regions of interest in the paraffin-embedded tissues, into one paraffin “master” block in a precisely spaced, array pattern. Sections from this “master” block are cut using a microtome, set onto glass slides, stained and analysed as for standard microscopy slides. Each microarray block can be cut into 100-500 sections which can be subject to independent tests.
Unfortunately, however, TMA has several limitations. Firstly the results are averaged over the whole sample meaning that if only a subsection of cells concentrated in one section of the sample drives the disease, this can be missed; and secondly, the sample is small so in a heterogeneous cell population (such as in a tumour), important information may be missed.
It is particularly desirable to obtain information on the spatial patterning of the markers and cells or sub-cellular structures. This patterning gives an indication of the type of cell-cell interaction occurring, and can provide useful information with regard to the diagnosis or prognosis of a subject.
Currently available methodology does not, however, allow spatial patterning to be analysed with more than a handful of biomarkers. IHC techniques, for example, provide one image for each stain but whilst it is believed that cells with particular IHC profiles may interact, there are few quantitative methods available for investigating these hypotheses.
The above-mentioned problems are addressed by the present invention.